Fig. 4: Transition of metaphase to anaphase-like microtubule bundle organization.

A Schematic top, cross section and side view of a flow chamber with a top reservoir. B TIRF microscopy images showing microtubule bundles with antiparallel overlaps that were pre-organized for ~5 min in the presence of 20 nM CDK1-phosphorylated PRC1-Alexa546 (green), 10 nM KIF4A-mGFP (blue) and 18 µM Alexa647-tubulin (red), followed by addition of 500 pM lambda-protein phosphatase (λ-PP), 1 mM MnCl₂ and 500 µM RO-3306 to the top reservoir to deliver the phosphatase to the imaging chamber to dephosphorylate PRC1. Times in min:sec, scale bars as indicated. C Exemplary snap shot depicts the temporal progression of a simulated antiparallel bundle of 4 microtubules connected by PRC1 (green) and KIF4A (blue), undergoing a transition from metaphase-to-anaphase-like organization. Microtubule orientation is indicated by color (plus ends up: cyan, plus ends down: red). The unbinding rate of PRC1 (k_off) and the microtubule plus end growth speed (v_g) were changed as indicated to trigger reorganization. D Quantification of average antiparallel overlap length of 10 experimental (top) and 10 simulated (bottom) microtubule bundles as a function of time. The gray dotted lines indicate the time of addition of λ-PP in the experiment, or change of unbinding rate (k_off) and microtubule growth speed in the simulation, as indicated. n = 2 independent experiments, total of 10 antiparallel overlaps (top), and n = 10 independent simulations, total of 10 antiparallel overlaps (bottom). Black and gray lines are mean and individual overlap length, respectively. Source data are provided as a Source Data file.